Ribosome profiling (Ribo-seq) can potentially provide detailed information about ribosome position on transcripts and estimates of protein translation levels in vivo. Hsp90 chaperones, which play a critical role in stress tolerance, have characteristic patterns of differential expression under nonstressed and heat shock conditions. By analyzing published Ribo-seq data for the Hsp90 chaperones in S. cerevisiae, we find wide-ranging artifacts originating from "multimapping" reads (reads that cannot be uniquely assigned to one position), which constitute ~25% of typical S. cerevisiae Ribo-seq datasets and ~80% of the reads from HEK293 cells. Estimates of Hsp90 protein production as determined by Ribo-seq are reproducible but not robust, with inferred expression levels that can change 10-fold depending on how multimapping reads are processed. The differential expression of Hsp90 chaperones under nonstressed and heat shock conditions creates artificial peaks and valleys in their ribosome profiles that give a false impression of regulated translational pausing. Indeed, we find that multimapping can even create an appearance of reproducibility to the shape of the Hsp90 ribosome profiles from biological replicates. Adding further complexity, this artificial reproducibility is dependent on the computational method used to construct the ribosome profile. Given the ubiquity of multimapping reads in Ribo-seq experiments and the complexity of artifacts associated with multimapping, we developed a publicly available computational tool to identify transcripts most at risk for multimapping artifacts. In doing so, we identify biological pathways that are enriched in multimapping transcripts, meaning that particular biological pathways will be highly susceptible to multimapping artifacts.

中文翻译：

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多重映射混淆了核糖体谱分析：Hsp90分子伴侣的案例研究。

核糖体概况分析（Ribo-seq）可能提供有关转录本上核糖体位置的详细信息以及体内蛋白质翻译水平的估计。Hsp90分子伴侣在胁迫耐受性中起着至关重要的作用，在非胁迫和热激条件下具有差异表达的特征模式。通过分析已公布的酿酒酵母中Hsp90伴侣的Ribo-seq数据，我们发现了来自“多重映射”读数（无法唯一分配给一个位置的读数）的广泛伪影，占典型酿酒酵母的25％。 Ribo-seq数据集和约80％的HEK293细胞读数。由Ribo-seq确定的Hsp90蛋白产量的估计是可重现的，但并不可靠，其推断的表达水平可能会改变10倍，具体取决于多重映射读段的处理方式。Hsp90分子伴侣在无压力和热休克条件下的差异表达在其核糖体图谱中产生了人工峰和谷，从而给人以错误的印象，称其为调节性翻译暂停。确实，我们发现多重映射甚至可以从生物复制品中复制出Hsp90核糖体概况的形状。进一步增加了复杂性，这种人工再现性取决于用于构建核糖体谱的计算方法。鉴于Ribo-seq实验中普遍存在多重映射读物以及与多重映射相关的人工产物的复杂性，我们开发了一种公共可用的计算工具来识别最易遭受多重映射人工产物风险的转录本。这样一来，我们就可以确定在多重映射转录本中丰富的生物学途径，